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Home , Bacteroidales, Klebsiella, Klebsiella oxytoca, Klebsiella pneumoniae, Neisseriaceae, Porphyromonas

and Providencia emerge as lone survivors following long-term starvation of oral microbiota

Jonathon L. Bakera, Erik L. Hendricksonb, Xiaoyu Tanga, Renate Luxc, Xuesong Hed, Anna Edlunda, Jeffrey S. McLeanb, and Wenyuan Shid,1

aGenomic Medicine Group, J. Craig Venter Institute, La Jolla, CA 92037; bDepartment of Periodontics, School of Dentistry, University of Washington, Seattle, WA 98195; cDepartment of Oral Biology, School of Dentistry, University of California, Los Angeles, CA 90095; and dDepartment of Microbiology, The Forsyth Institute, Cambridge, MA 02142

Edited by Jeff F. Miller, University of California, Los Angeles, CA, and approved March 20, 2019 (received for review December 3, 2018) It is well-understood that many have evolved to survive acids as a source of carbon and energy (4). Therefore, it was catastrophic events using a variety of mechanisms, which include likely that in a complex multispecies community, a succession of expression of stress-response genes, quiescence, necrotrophy, and and strains would increase and decrease in relative metabolic advantages obtained through mutation. However, the abundance throughout the course of the experiment, based on dynamics of individuals leveraging these abilities to gain a compet- which species were most fit in the changing environment. itive advantage in an ecologically complex setting remain unstudied. To our knowledge, there have been no previous reports on the In this study, we observed the saliva microbiome throughout the interplay of a complex community of human-associated bacteria ecological perturbation of long-term starvation, allowing only the subjected to long-term starvation. To begin to address this knowl- species best equipped to access and use the limited resources to edge gap, the present study monitored a saliva-derived complex survive. During the first several days, the community underwent a oral microbiota during starvation in saline solution and saliva for death phase that resulted in a ∼50–100-fold reduction in the num- 100 d. The bacterial community residing in the human oral cavity is ber of viable cells. Interestingly, after this death phase, only three a particularly unique microbiota that undergoes cyclical expansions species, , , and Providen- and contractions of microbial diversity as a result of both host food cia alcalifaciens, all members of the family , ingestion intervals and hygienic practices, such as brushing and use

appeared to be transcriptionally active and recoverable. Klebsiella of mouthwash. These perturbations respectively result in cycles of MICROBIOLOGY are significant human , frequently resistant to multiple relative feast or famine and regular killing or removal of large , and recently, ectopic colonization of the gut by oral swaths of the microbial population, after which ecological succes- Klebsiella was documented to induce dysbiosis and inflammation. sion begins anew (10). Dental caries and periodontitis represent MetaOmics analyses provided several leads for further investiga- two extremely prevalent and costly diseases that are now rec- tion regarding the ecological success of the Enterobacteriaceae. ognized as the result of localized ecological catastrophes of the The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural Significance products closely resembling cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacter- This study illustrates the dynamics of the oral microbiome iaceae persist much longer than their more benign neighbors in the during long-term starvation. After an initial ecological collapse, salivary microbiome when faced with starvation. This is particularly only three species were recoverable and displayed significant significant, given that surfaces contaminated with oral flu- transcriptional activity: Klebsiella pneumoniae, Klebsiella oxy- ids, especially sinks and drains, are well-established sources of out- toca, and Providencia alcalifaciens. Klebsiella spp. are signifi- breaks of drug-resistant Enterobacteriaceae. cant human pathogens and are frequently resistant to multiple classes of antibiotics. In addition to its status as a clinical oral microbiome | microbial ecology | Klebsiella scourge in its own right, K. pneumoniae has emerged as a chief facilitator in the transfer of genes from the any bacteria are well-equipped to deal with exposure to environment to pathogens. Hospital surfaces contaminated Madverse environmental events such as starvation, oxidative with oral fluids are well-documented sources of outbreaks of stress, and , as well as fluctuations in temperature, drug-resistant Enterobacteriaceae; therefore, the ability of pH, and osmolality. Diverse strategies for coping with these Klebsiella to outcompete its neighbors during starvation and stresses have evolved, including expression of stress response survive long-term in saliva is particularly noteworthy. genes (1), quiescence (2), necrotrophy (3), and growth advan- tages gained through mutation (4). Although these systems are Author contributions: J.L.B., X.H., J.S.M., and W.S. designed research; J.L.B., E.L.H., X.T., A.E., and J.S.M. performed research; J.L.B., E.L.H., R.L., X.H., A.E., J.S.M., and W.S. analyzed increasingly understood, little is known regarding the dynamics data; and J.L.B. wrote the paper. of individual species leveraging these abilities to gain a com- Conflict of interest statement: J.L.B. is a part-time consultant for uBiome, Inc. W.S. is a petitive advantage in an ecologically complex setting. In the case part-time chief science officer of C3J Therapeutics, Inc., which has licensed technologies of long-term starvation, the bulk of research on bacterial dy- from the University of California Regents that could be indirectly related to this namics and survival mechanisms has been performed using research project. monospecies cultures of (4–9). In E. coli, pop- This article is a PNAS Direct Submission. ulations exhibited a death phase at ∼3 d of starvation, resulting This open access article is distributed under Creative Commons Attribution-NonCommercial- in a loss of >99% of the population (4). This death phase was NoDerivatives License 4.0 (CC BY-NC-ND). followed by a long-term stationary phase, in which viable cell Data deposition: The raw sequencing files used in the genomic, metagenomic, and tran- scriptomic analyses in this study have been deposited in the Sequence Read Archive, https:// counts plateau virtually indefinitely (i.e., for years) (4). During www.ncbi.nlm.nih.gov/sra [accession nos. PRJNA525688 (genomes) and PRJNA525517 the long-term stationary phase, various subpopulations carrying (metagenomes/transcriptomes)]. advantageous mutations (growth advantage in stationary phase 1To whom correspondence should be addressed. Email: [email protected]. [GASP] mutants) arose and came to dominate the culture, dis- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. placing their less-fit siblings (4). These mutations frequently 1073/pnas.1820594116/-/DCSupplemental. resulted in increased ability to catabolize one or more amino Published online April 11, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1820594116 PNAS | April 23, 2019 | vol. 116 | no. 17 | 8499–8504 Downloaded by guest on September 26, 2021 oral microbiome (11–14). One of the major impediments to the study AB9 of complex, human-associated microbial communities is the difficulty 10 PBS:saliva 7 *** cultivating such diverse ecologies in a well-controlled laboratory 108 PBS setting. The oral microbiota was chosen as a model system for this 107 6 pilot study because of the existence of a well-established in vitro 106 CFU/ml 5 culture system using media that allows for the growth of a diversity of 105 Shannon Index 4 species approaching that of an in vivo human mouth (15). 104 PBS:saliva PBS 050100 Time (days) 84 Results CDPBS:saliva 72 7 PC2 (23.8%) PBS 59 Long-Term Starvation of a Complex Oral Community Results in a **** 45 32 Death Phase, Followed by a Long-Term Stationary Phase. The start- 6 20 12 ing community in this study was derived from a previous study 4 5 2

that optimized media (SHI media) and growth conditions to Days of Starvation 1 stably maintain the highest diversity of oral bacteria achievable Shannon Index 0 4 PC1 (51.4%) 0 in vitro to date (15). The community was generated from the 1 2 4 12 20 32 45 59 72 84 PC3 (3.7%) pooled saliva of six healthy adults, had a microbial diversity Days of Starvation PBS:saliva PBS:saliva outgrowth approaching that of human oral cavity, and responded to a E starving community in fresh media carbohydrate pulse in a manner similar to in vivo dental plaque Days of Starvation 0 1 4 12203245597284 0 4 12 32 59 84 (15). After overnight batch growth in SHI media, the community Proteus Enterobacteriacae; other was harvested and subsequently starved in aliquots of either PBS Kluyvera or a 1:1 mixture of PBS and cell-free saliva. Reminiscent of Klebsiella Enterobacter

monospecies cultures of E. coli (4), the communities (both in Klebsiella Other PBS and PBS:saliva) first experienced a death phase with a rapid Fusobacterium decrease in colony-forming units per milliliter over time, fol- Veillonella Peptostreptococcus lowed by a stabilization, a long-term stationary phase (Fig. 1A). Parvimonas Stomatobaculum It is important to note that many taxa that are viable in the liquid Streptococcus

culture of the community cannot grow as isolated colonies on Granulicatella Streptococcus Gamella solid media, and thus are not measured in Fig. 1A. Therefore, although the colony-forming unit measurement is useful for il- lustrating the overall trend of viable cells in the community, the Mobiluncus results are likely an underestimate, making it critical to com- Corynebacterium

plement this assay with mRNA-based taxonomic profiling, as PBS PBS outgrowth described below, to determine the living members of the com- F starving community in fresh media munity during starvation. Illumina sequencing of 16S rDNA V3- Days of Starvation 01 412203245597284 0 4 12 32 59 84 V4 amplicons revealed that alpha diversity was higher in the Proteus Enterobacteriacae; other PBS:saliva community than the PBS community, reflective of the Kluyvera supplementary substrates provided by the sterilized saliva at the Klebsiella Enterobacter start of the starvation (Fig. 1B). Intrasample diversity (alpha Neisseria Other diversity) of the community increased across the first several days Fusobacterium of starvation and then decreased to levels similar to that of the Veillonella Peptostreptococcus starting community after day 32 (Fig. 1C). Intercommunity di- Parvimonas Stomatobaculum versity (beta diversity) also shifted over time, particularly during Streptococcus the first 12 d (Fig. 1D), followed by relative stabilization. The Granulicatella Gamella presence of saliva in the starvation medium also had an effect on Prevotella Alloprevotella the community, as the two starvation communities separated in Porphyromonas Principal Coordinates Analysis (PCoA) space over time (Fig. Bacteroidales Mobiluncus 1D). There were shifts in the relative abundances of many taxa, Corynebacterium with by far the most notable change being an increase in the number of Enterobacteriaceae, particularly Klebsiella, at the Fig. 1. Shifts in diversity and relative abundances of constituents of the expense of Streptococci, which had been the most abundant saliva microbiome during long-term starvation. (A) Number of viable cells in genera in the starting communities (Fig. 1 E and F). Because the the community, as determined by colony-forming units during starvation in PBS:saliva or PBS over the course of 100 d. (n = 3). (B) Alpha diversity of the DNA harvested from the starving community was likely to con- < tain the DNA of dead cells, aliquots of the starvation culture PBS:saliva or PBS communities across 84 d of starvation. ***P 0.001, unpaired t test. (C) Alpha diversity of the PBS:saliva and PBS starvation communities over from various points were used to inoculate fresh SHI media, and the course of 84 d of starvation. ****P < 0.0001, Brown-Forsythe test. (D) the outgrowth culture was investigated with 16S rDNA sequencing “ ” PCoA plot of unweighted UNIFRAC distances illustrating beta diversity of the after overnight growth to observe the live species from the initial PBS:saliva and PBS communities over the course of 84 d of starvation. (E) starvation experiment (Fig. 1 E and F). The increase in Enter- Relative abundances of bacterial genera in the PBS:saliva starvation exper- obacteriaceae 16S rDNA observed during long-term starvation iment, and the starvation community after overnight outgrowth in fresh was much more dramatic in the outgrowth communities. After only media, as determined by Illumina sequencing of 16S amplicons. (F) Relative 4 d of starvation, the proportion of Klebsiella in the communities abundances of bacterial genera in the PBS starvation experiment, and the had increased dramatically, and by day 12, Enterobacteriaceae starvation community after overnight outgrowth in fresh media, as determined accounted for more than 90% of the relative abundance of the by Illumina sequencing of 16S amplicons. 16S rDNA in the outgrowth communities (Fig. 1 E and F). Al- though Klebsiella was the genus with the highest relative abundance, species of “unclassified” Enterobacteriaceae (highly likely to be P. After 20 d of Starvation, Only Enterobacteriaceae Are Recoverable on alcalifaciens, as described below) increased in relative abundance Solid Media. When plated on SHI media agar, the day 0 com- during the later points of the outgrowth. The community starving munity produced colonies with a wide variety of morphologies, in PBS underwent a transition similar to that of the community reflective of the diversity of species present (Fig. 2). During the starving in PBS:saliva, albeit with a reduced abundance of Neisseria death phase, the relative number of the large mucoid colonies and Porphyromonas (Fig. 1F), indicating that components in the obtained increased dramatically, such that by day 20, and all filtered saliva made these genera more competitive. following times, all the colonies obtained were large and mucoid

8500 | www.pnas.org/cgi/doi/10.1073/pnas.1820594116 Baker et al. Downloaded by guest on September 26, 2021 Day 0 Day 4 Day 20 Day 84 Day 100

Fig. 2. Colony morphology during long-term starvation. Representative image of the PBS:saliva community on SHI agar after the indicated number of days of long-term starvation.

(Fig. 2). A selection of diverse colony morphologies across three P. alcalifaciens strains that increased in prevalence to >95%, in times was isolated and identified, using Sanger sequencing of full- both PBS and PBS:saliva long-term starvation communities length 16S rDNA amplicons (Dataset S1). At day 1, several species (Dataset S3). In K. pneumoniae, these SNPs occurred in the topB of Streptococcus were recovered, as were Gamella sanguinis, Kleb- topoisomerase, the allS_2 LysR family transcriptional regulator, siella pneumoniae,andKlebsiella oxytoca. At day 20, K. pneumoniae, the glyS glycine tRNA ligase, and the lamB_1 maltoporin. Most K. oxytoca, Enterobacter homaechei,andProvidencia alcalifaciens intriguingly, both topoisomerase and LysR-type regulators were were the only recoverable species. Finally, at day 84 and day 100, previously identified as GASP alleles, although the mechanism of only K. pneumoniae and P. alcalifaciens were recoverable under the the contribution of mutations in these genes to the GASP phenotype conditions tested. This is a particularly interesting result because all remains unknown (6, 21). Maltoporin, an outer membrane trans- the surviving organisms are documented pathogens and are fre- porter of maltose, has been shown to be critical during adaptation to quently drug resistant (16–18). new growth conditions (22), and thus likely represents a GASP allele. In P. alcalifaciens, two SNPs that increased in frequency during long- MICROBIOLOGY RNA Sequencing Confirms That Enterobacteriaceae Are the Dominant term starvation were located in the tamB component of the TAM (and Likely Only Living) Community Members After Long-Term translocation and assembly module and in Toxin B (toxB). In- Starvation. To obtain a finer resolution of the viable and ac- terestingly, tamB mutants have significantly decreased virulence in K. tively transcribing species, shotgun sequencing of cDNA from pneumoniae and (23, 24). Although it is cur- the transcriptome of the community at five points during the rently unclear whether these SNPs contributed to the success of long-term starvation was performed, followed by Metaphlan2 these Enterobacteriaceae during long-term starvation, the fact that analysis to calculate the relative abundances of taxa (19) (Fig. 3). these variants increased in frequency several times, independently, In the PBS:saliva starvation community, Firmicutes, namely, the highlights a need for further investigation. genera Streptococcus, Gamella, Peptostreptococcus, and Veillo- nella, represented >90% of the RNA at day 0. At day 4, there Enterobacteriaceae Exhibit Shifts in the Transcriptome During Long- was a diversity of RNA from Streptococcus, Peptostreptococcus, Term Starvation. To obtain information about transcriptional ac- Neisseria, Veillonella, Klebsiella, Porphyromonas, and Fusobacte- tivity among the Enterobacteriaceae during the long-term starva- rium. By day 20, Klebsiella increased in number such that the tion, the cDNA Illumina sequencing reads were mapped to the genus represented 46% of all RNA. This increase was largely at assembled genomes of K. pneumoniae, K. oxytoca,andP. alcalifa- the expense of Firmicutes, which decreased to <3% of RNA. ciens. Genes that were differentially abundant between day 0 and By day 84, Providencia accounted for 69% of the RNA, whereas subsequent times were identified using DeSeq2 (Dataset S4). Gene Neisseria accounted for 20%. Ultimately, after 100 d of starva- ontology terms for each differentially abundant gene were sum- tion, Providencia accounted for 90% of the RNA, indicating that it marized and visualized using Revigo (25) and a custom R script (SI was the major living organism remaining in the community. As with Appendix,Fig.S1). Eleven, 14, 15, and 5 nonredundant biological the 16S rDNA profiling, the changes in abundance of taxa in the processes exhibited increased expression at days 4, 20, 84, and 100 PBS community were similar to the major trends of the PBS:saliva compared with day 0, respectively. Meanwhile, 22, 10, 20, and 40 community. Dataset S2 contains Krona (20) visualizations of Met- nonredundant biological processes exhibited decreased expression aphlan2 analysis of the transcriptome from both PBS:saliva and on days 4, 20, 84, and 100, respectively. Interestingly, day 20, the PBS communities, as well as Metaphlan2 analysis of metagenomes point at which Enterobacteriaceae had become the most abundant (DNA) from both communities. Collectively, the above analyses members of the community, was the only observed point at which indicate that species other than Enterobacteriaceae quickly lose the number of biological processes with increased expression viability and are no longer transcriptionally active after ∼10 d of exceeded the number of biological processes with decreased ex- long-term starvation. K. pneumoniae quickly becomes the most pression. Negative regulation of flagellum , negative regu- abundant member of the community, based on RNA, early during lation of biofilm formation, and positive regulation of carbohydrate the long-term stationary phase, but is overtaken by P. alcalifaciens metabolic processes were the biological processes with the most by days 84 and 100. Notably, ∼10 d is also the length of time re- highly elevated expression at days 20, 84, and 100 compared quired for starved cultures of E. coli to develop GASP mutations with day 0. By day 100, these three biological processes, along with that allow them to displace wild-type siblings (4). positive regulation of catalytic activity, and oxidation-reduction process were the only biological processes that were up-regulated Whole-Genome Sequencing of Enterobacteriaceae Isolates Reveals compared with day 0. This may suggest that the Enterobacteriaceae Single Nucleotide Polymorphism Accumulation During Starvation. may be attempting to conserve energy and passively transport to DNA obtained from Enterobacteriaceae isolates from days 0, a location with a novel food source. Xylose metabolism, valine 20, and 84 (Dataset S1) was subjected to Illumina shotgun se- biosynthetic process, and xylose transport were the three most quencing. Genomes were assembled from each point and com- highly up-regulated biological pathways at day 4, indicating that pared with the earliest available point for each species (in the these pathways may be important to the Enterobacteriaceae early PBS community). There were six nonsynonymous single nucle- during long-term starvation, and that they may play a role in otide polymorphisms (SNPs) observed in the K. pneumoniae and adapting to new conditions. Collectively, pathway analysis provides

Baker et al. PNAS | April 23, 2019 | vol. 116 | no. 17 | 8501 Downloaded by guest on September 26, 2021 Day 0 Day 4 Day 20 Providencia unclassified 1% Klebsiella Neisseria 3% Peptostreptococcus oxytoca 11% unclassified 24% 21% Neisseria unclassified 43% Klebsiella unclassified Peptostreptococcus Neisseria unclassified 2% Neisseria Klebsiella Clostridiales oxytoca 3% Neisseriaceae Firmicutes 2% Peptostreptococcus stomatis 5% Parvimonas Klebsiella Species unclassified Species ProteobacteriaSpecies 11% Porphyromonas Gemella

pneumoniae 7% Klebsiella Gemella 3% Veillonella atypica Firmicutes gingivalis

Bacteroidetes unclassified 4% Bacteroidales Klebsiella

Enterobacteriaceae Bacilli Porphyromonas

pneumoniae 33% Bacteroidales Porphyromonas Lactobacillales Veillonella Gemella 16% Veillonella Veillonella 5% Fusobacterium 34% Porphyromonas 18% Bacteroidetes sanguinis 4% Streptococcus unclassified periodonticum endodontalis oral taxon 274 unclassified 6%

2% Neisseria unclassified 2 more

Veillonella Granulicatella 8 more

1% Fusobacterium atypica 3% Eubacterium unclassified 4% Bacteroidetes 0.7% Actinobacteria <1%

periodonticum infirmum 2% Actinobacteria <1% Fusobacterium 0.3% Mycoplasma hominis <1% Mycoplasma hominis <1% Peptostreptococcus unclassified 4% Streptococcus Actinobacteria <1% anginosus 5% Mycoplasma hominis <1%

Streptococcus

parasanguinis 7% Day 84 Day 100 Streptococcus salivarius 1% 1% Moraxellaceae

20% Neisseria Providencia unclassified unclassified 89% Neisseria Neisseriaceae

1% Providencia alcalifaciens Proteobacteria Proteobacteria 2% Neisseria flavescens 3% Neisseria unclassified 1% Neisseria flavescens Providencia 2% Porphyromonas Species endodontalis Species 1% Porphyromonas endodontalis unclassified 69% 4% Klebsiella pneumoniae 4% Klebsiella pneumoniae

Klebsiella 2 more

Klebsiella Gammaproteobacteria Enterobacteriaceae Enterobacteriaceae Providencia Providencia

Fig. 3. Relative abundances of actively transcribing taxa in the PBS:saliva long-term starvation community. At the indicated day based on Metaphlan2 analysis of mRNA.

leads for further research into the mechanism behind the success spectral clusters with a GNPS library hit to a cyclic depsipeptide of the Enterobacteriaceae during long-term starvation. with a parent mass of 1,124.6 and a cyclic peptide sequence of MeGlu-O-ξIle-Phe-Pro-Gly-MeVal-MeGlu-ξIle-Pro-Val (Fig. 4 B Natural Products Analysis Indicates Generation of Cyclic Depsipeptides and C). Cyclic depsipeptides are a fascinating class of small mole- by Enterobacteriaceae. To explore the dynamics of the small mole- cules that frequently have antimicrobial and/or anticancer activity cules produced by the communities during long-term starvation, and are the subject of ongoing research (28–32). These two spectral harvested cells, as well as culture supernatants, of both the com- clusters networked with two other unidentified spectral clusters munity and the isolated strains were analyzed by liquid chroma- with a parent mass of 1,025.5, which may indicate loss of a valine tography mass spectrometry, followed by spectral analysis using residue from the structure of the known cyclic depsipeptide (Fig. Global Natural Products Social Molecular Networking (GNPS) 4B). All four cyclic depsipeptide spectral clusters appeared to be (26). As is the present case with most metabolomics analysis, the associated with Enterobacteriaceae, based on the number of spec- majority of MS spectra were unannotated/unknown, with only tra associated with single-species isolates. This family of molecules ∼40% of the spectral clusters mapping to known annotations. also appeared in the community samples during early times, when Twenty-five unknown spectral clusters were assigned a putative the main shift in species abundance was occurring (∼day 4; Fig. molecule class based on molecular networking analysis. The com- 4A). These molecules may be bactericidal compounds secreted plete molecular network was visualized using Cytoscape (27) and is by the Enterobacteriaceae to kill the neighboring species during available in the SI Appendix,Fig.S3. A large number of the MS starvation for use in necrotrophy, and further study of these compounds spectra that networked with known lipid spectra were found only in is warranted. the cell pellets, congruent with the concept that these are membrane-associated molecules and are unlikely to be secreted. Enterobacteriaceae Increase in Abundance During Starvation in There were a large number of MS spectra that only appeared in the Additional Communities from Individual Donors. To ensure that community, either because the species synthesizing them were not the phenomenon of the Enterobacteriaceae species becoming species that were isolated or because isolates that were analyzed did the dominant, living members of the starving community was not not make the natural products in a single-species culture. There unique to this consortium of bacteria, the same long-term star- were more spectra associated with the Enterobacteriaceae vation experiment (using PBS as starvation media) was performed species than the Streptococci, which could be expected given that on five additional salivary communities, each isolated from the the Enterobacteriaceae quickly increased in relative abundance saliva of a single, healthy individual. 16S rDNA PCR-denaturing in the communities during starvation. Interestingly, valine was sig- gradient gel electrophoresis was used to monitor taxonomic pro- nificantly more abundant at later points (Fig. 4A and SI Appendix, files of the five communities during the first 20 d of starvation (SI Fig. S2), in agreement with up-regulation of transcription of valine Appendix, Fig. S4). The denaturing gradient gel electrophoresis biosynthetic processes among the surviving Enterobacteriaceae (SI profiles of Community numbers 1 and 5 contained bands repre- Appendix,Fig.S1; day 20). The GNPS Dereplicator identified two senting Enterobacteriaceae, as determined by Sanger sequencing of

8502 | www.pnas.org/cgi/doi/10.1073/pnas.1820594116 Baker et al. Downloaded by guest on September 26, 2021 A Number of Spectra 02040 CP Super.Entero. Strep. y 72 y 59 y 84 y 100 Family a a ay 20 a ay 12 Solubility Bacterial Day 10 Day D D Day 32 Day 45 Day D Day 0 Day 1 Day 2 Day 4 Day 6 Day 8 Day D Da D 115.033 L−VALINE L−PHENYLALANINE 115.034 Methionine L−VALINE 89.107 Isoleucine Amino Acids Acetyl−ornithine acid 176.068 150.056 DL−Phenylalanine L−Tryptophan L−Histidine Methionine Cyclic 1025.54 1024.62 Depsipeptides 1024.61 1025.55

H BCO

O 100% 584.3 344.18

O 70.06 H N 1025.54 1124.61 N 199.12 171.15 H

O 927.49 N 118.08 810.41 H 50%

N N O 782.45 H O Intensity 455.25 O N O O N 683.38 O 654.39 326.16 1008.54 1221.44 1150.84 1200.68 1281.88 427.27 1373.47 1352.55 1432.65 1470.53 1532.05 1566.81 1074.69 1090.12 1616.89 1692.10

H 405.70 526.28 O H 870.41 N O 0% N

O OO H

1025.55 1124.62 MICROBIOLOGY MeGlu-O-ξIle-Phe-Pro-Gly-MeVal-MeGlu-ξIle-Pro-Val 200 400 600 800 1000 1200 1400 1600 m/z

Fig. 4. Accumulation of valine and production of cyclic depsipeptides during long-term starvation. (A) Heat map illustrating the number of liquid chromatography mass spectrometry spectra associated with the indicated spectral cluster after the indicated number of days of starvation. Spectral clusters are grouped into molecule classes (e.g., amino acids) based on molecular networking to GNPS library hits. Row clustering within molecule classes was performed using a Pearson similarity distance matrix. Spectral clusters are named using the GNPS library hit where applicable; otherwise, clusters are named using the liquid chromatography mass spectrometry parent mass. The Solubility column indicates the percent of the spectra (0–100%) within a cluster that were associated with the samples extracted from the culture supernatant (Super.) versus the cell pellet (CP). The Bacterial Family column indicates the percentage of spectra within a cluster that were associated with samples extracted from isolates of Streptococcaceae (Strep.) versus Enterobacteriaceae (Entero.). Only amino acids and cyclic depsipeptides are shown here; the full heat map with all spectral clusters is presented in the SI Appendix,Fig.S2.(B) Molecular subnetwork of the four cyclic depsipeptide spectral clusters. Node size corresponds to the total number of spectra in each cluster. Node label indicates parent mass. Edge width indicates the cosine scorebetween spectral cluster nodes. Black outlines denote nodes which mapped to the GNPS library hit, cyclo-MeGlu-O-ξIle-Phe-Pro-Gly-MeVal-MeGlu-ξIle-Pro-Val, with indicated chemical structure. The complete molecular network is available in the SI Appendix,Fig.S3.(C) Comparison of MS/MS spectra from spectral cluster with parent mass 1,124.61 (black bars) to GNPS library spectrum for cyclo-MeGlu-O-ξIle-Phe-Pro-Gly-MeVal-MeGlu-ξIle-Pro-Val (green bars).

the DNA contained within the excised band. Most importantly, the provides a reservoir for would-be intestinal pathogens, such as density of the Enterobacteriaceae bands increased during starva- Klebsiella (33). Furthermore, aside from being a substantial path- tion, concurrent with a decrease in the density of most other bands ogen in its own right, evidence is accumulating that K. pneumoniae within the denaturing gradient gel electrophoresis profile. This serves as a key trafficker of drug resistance loci from the environ- finding signifies an increase in the relative abundance of Enter- ment to human pathogens (34). obacteriaceae at the expense of other taxa, indicating that the The mechanisms employed by these Enterobacteriaceae to outlast phenomenon observed in the main experiment was not exclusive their neighbors during long-term starvation await investigation. The to that starting community of bacteria. The other three communities Enterobacteriaceae encode among the largest genomes in the oral did not appear to have significant numbers of Enterobacteriaceae at microbiome (35) and, as such, have added metabolic flexibility any point (SI Appendix,Fig.S4). compared with Streptococci and other common constituents of the oral cavity (36). It is likely that during long-term starvation, species Discussion with reduced genomes have less metabolic flexibility and are at a This study provides an account of a complex, human-associated significant disadvantage to Enterobacteriaceae (37). Klebsiella are microbial community experiencing the ecological perturbation of , and all Enterobacteriaceae are capable of using nitrate, long-term starvation. The finding that Klebsiella and Providencia S-oxides, and N-oxides as terminal electron acceptors (36). Thus, the species were the apparent sole survivors in a community after long- concept that these abilities were advantageous during long-term term starvation is significant and highly intriguing. K. pneumoniae, K. starvation remains an attractive hypothesis. In addition, the oxytoca,andP. alcalifaciens are all members of the Enter- MetaOmics analyses performed in this study provided several obacteriaceae family of Proteobacteria. K. pneumoniae is a significant additional hypotheses for further investigation. The increased abun- and represents the ‘K’ in the ESCKAPE pathogens, a dance of SNPs in several genes in K. pneumoniae and P. alcalifaciens group of organisms frequently resistant to multiple antibiotics (16). may represent GASP mutations, which were originally discovered K. oxytoca and P. alcalifaciens are also opportunistic pathogens and in E. coli, another member of the family Enterobacteriaceae (4). are frequently drug resistant (17, 18). Oral K. pneumoniae was re- Overall analysis of the Enterobacteriaceae transcriptome cently shown to induce inflammation and dysbiosis in the gut after indicated that the species may be attempting to conserve energy and ectopic colonization, and it was hypothesized that the oral cavity use passive transport to locate a novel food source. Meanwhile,

Baker et al. PNAS | April 23, 2019 | vol. 116 | no. 17 | 8503 Downloaded by guest on September 26, 2021 several intriguing cyclic depsipeptides may have been employed by aerosol formation after subsequent use of the sink leads to spread of the Enterobacteriaceae to kill their neighbors. the . This danger is compounded by the frequent horizontal The results presented here also illustrate the value of RNA-based gene transfer of resistance genes employed by Enterobacteriaceae detection methods. Although a large number of taxa were present at (34, 39). Elucidation of the mechanisms used by the Enter- all points, according to sequencing of 16S amplicons, as well as met- obacteriaceae to survive long-term starvation in a community setting agenomes, sequencing of mRNA revealed a much more drastic re- is highly important, and further research is currently in progress. duction of species during and after the death phase. This loss of diversity was also reflected in the sequencing of the outgrowth com- Materials and Methods munities and the plating assay, from which only the three Enter- More detailed methods with additional references are available in the SI Appendix. obacteriaceae species were recoverable at day 20 under the conditions The starting bacterial community (S-mix), derived from the saliva of six healthy tested, despite the presence of DNA from a multitude of species in the subjects, ages 25–35 y, has been described previously (15). After overnight growth in

community at that time. Because some bacteria are known to enter a SHI medium in microaerophilic conditions (2%O2,5%CO2, 93%N2), 1 mL aliquots of viable but not culturable state during adverse growth conditions, the S-mix were starved in either 1× PBS or a 1:1 mixture of 1× PBS and cell-free saliva. lack of taxonomic diversity at the transcriptional level during the later Colony-forming units per milliliter was determined by growth for 72 h on SHI media points of starvation serves as an important validation that the colony- agar under microaerophilic conditions. 16S rDNA taxonomic profiling was per- forming units per milliliter and recoverable species on solid media formed by Illumina sequencing of V2-V4 amplicons, followed by analysis using reported here are not largely underestimated. QIIME. Metagenomes were assembled de novo using SPAdes and aligned using The ability of the Enterobacteriaceae to survive longer than other Mauve. Metatranscriptomic reads were mapped to Enterobacteriaceae genomes members of the saliva microbial community may have a great deal of using Burrows-Wheeler alignment, and genes that were significantly differen- clinical significance. Although the long-term starvation model used in tially expressed were identified using DeSeq2. Natural products analysis was this study is unlikely to simulate the oral cavity, where periods of performed using reverse-phase high-pressure liquid chromatography followed starvation are much shorter, it is presumably analogous to the suc- by tandem mass spectrometry, as detailed in the SI Appendix. Raw sequencing cession that occurs when human saliva is deposited in environmental data have been deposited in the Sequence Read Archive (SRA) (40, 41). locations not exposed to rapid desiccation. Contaminated hospital surfaces, particularly sinks, have been the source of outbreaks of ACKNOWLEDGMENTS. The authors thank Roberta Faustoferri for helpful proofreading of the manuscript. This study was supported by National Institutes multidrug-resistant Klebsiella (17, 38, 39). It is therefore easy to of Health/National Institute of Dental and Craniofacial Research Grants F32- imagine a scenario in which Enterobacteriaceae survive for extended DE026947 (to J.L.B.), R00-DE024543 (to A.E.), and R01-DE020102 and R01- periods in mixtures of saliva and water in sinks and drains, where DE026186 (to X.H., J.S.M., and W.S.).

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